Immuno-localisation studies of ISP1 and ISP3 defines apical distribution. A) Localisation of ISP1-GFP by immuno-electron microscopy. i. part of a zygote showing a protrusion with underlying dense material labeled with gold particles. ii. Low power view of the apical end of an ookinete showing gold particles associated with the plasma membrane/IMC. Note there are more particles on one side. M– micronemes. iii. Enlargement of the enclosed area in ii showing the increased number of gold particles associated with the plasma membrane/IMC. Scale bars are 100 nm. B) Co-staining of ISP1-GFP and ISP3GFP expressing ookinetes with antibodies to GFP (green) and IMC marker GAP45 (Red). C–D) Indirect immuno-fluorescence showing the co-localisation of ISP1-GFP and ISP3-GFP with respect to the cytoskeletal marker a-tubulin, during gametocyte and ookinete stages (Scale bar 5 5 mm). Strong co-localisation can be seen at the apical end for both ISP1 and ISP3, with a-tubulin (red). Nuclei were detected using Hoechst dye, and the cells were displayed by differential interference contrast (DIC). Merge is the composite of Hoechst, GFP and GAP45/tubulin. Scale bar 5 mm. Poulin B, Patzewitz EM, Brady D, Silvie O, Wright MH, Ferguson DJ, Wall RJ, Whipple S, Guttery DS, Tate EW, Wickstead B, Holder AA, Tewari R. Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite. Biol Open. 2013 2(11):1160-70. PMID: 24244852